Research Article

Garlic Constituent Diallyl Trisulfide Prevents Development of

Poorly Differentiated Prostate Cancer and Pulmonary
Metastasis Multiplicity in TRAMP Mice
1,2 1 1 1 2
Shivendra V. Singh, Anna A. Powolny, Silvia D. Stan, Dong Xiao, Julie A. Arlotti,
1 1 2 1
Renaud Warin, Eun-Ryeong Hahm, Stanley W. Marynowski, Ajay Bommareddy,
3 2
Douglas M. Potter, and Rajiv Dhir
1
Department of Pharmacology and Chemical Biology, and 2University of Pittsburgh Cancer Institute, University of Pittsburgh School of
Medicine, and 3Biostatistics Department, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania

Abstract usually diagnosed in the sixth or seventh decade of life providing a

Identification of agents that are nontoxic but can delay onset
and/or progression of prostate cancer, which is the second
leading cause of cancer-related deaths among men in the
United States, is highly desirable. We now show that p.o.
gavage of garlic constituent diallyl trisulfide (DATS; 1 and
2 mg/day, thrice/week for 13 weeks beginning at age 8 weeks)
significantly inhibits progression to poorly differentiated
prostate carcinoma and pulmonary metastasis multiplicity
in transgenic adenocarcinoma of mouse prostate (TRAMP)
mice without any side effects. There was a trend of a decrease
in average wet weights of the urogenital tract and prostate
gland in 1 and 2 mg DATS–treated mice compared with
controls (f25–46% decrease in DATS-treated mice compared
with controls). The incidence and the area of the dorsolateral
prostate occupied by the poorly differentiated carcinoma
were significantly lower in both 1 and 2 mg DATS–treated
mice compared with control mice. In addition, DATS
administration resulted in a statistically significant decrease
in pulmonary metastasis multiplicity compared with controls
(P = 0.002). The dorsolateral prostate from DATS-treated
TRAMP mice exhibited decreased cellular proliferation in
association with induction of cyclinB1 and securin protein
levels, and suppression of the expression of neuroendocrine
marker synaptophysin. However, DATS administration did not
have any appreciable effect on apoptosis induction, angio-
genesis, or natural killer and dendritic cell function. In
conclusion, the results of the present study show, for the first
time, that DATS administration prevents progression to
invasive carcinoma and lung metastasis in TRAMP mice.
[Cancer Res 2008;68(22):9503–11]
Introduction
Prostate cancer is one of the most commonly diagnosed visceral
malignancies and the second leading cause of cancer-related
deaths among men in the United States (1). Prostate carcinogenesis
is characterized by gradual transformation of normal prostate
epithelium to prostatic intraepithelial neoplasia (PIN), localized
tumor to advanced and metastatic disease (2). Prostate cancer is
Requests for reprints: Shivendra V. Singh, 2.32A Hillman Cancer Center Research
Pavilion, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-
7828; E-mail: singhs@upmc.edu.
I2008 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-08-1677
large window of opportunity for intervention to prevent or slow
progression of the disease. Consequently, identification of novel
agents that are nontoxic but can delay onset and/or progression of
human prostate cancer is highly desirable. Natural products have
received increasing attention in recent years for the discovery of
novel cancer preventive and therapeutic agents (3).
Epidemiologic studies continue to support the premise that
dietary intake of Allium vegetables (e.g., garlic) may lower the
risk of various types of malignancies including cancer of the
prostate (4–6). For example, the risk of prostate cancer was
shown to be significantly lower in men consuming >10 g/day of
total Allium vegetables than in men with total Allium vegetable
intake of <2.2 g/day in a population-based, case-control study (6).
The anticarcinogenic effect of Allium vegetables is attributable to
organosulfur compounds (OSC) including diallyl sulfide, diallyl
disulfide (DADS), and diallyl trisulfide (DATS), which are
generated upon processing (e.g., cutting or chewing) of these
vegetables (7). The OSCs have been shown to afford significant
protection against chemically induced cancers in animal models,
including benzo(a)pyrene-induced forestomach and pulmonary
carcinogenesis in mice, N-nitrosomethylbenzylamine–induced
esophageal cancer in rats, and azoxymethane-induced colon
carcinogenesis in rats (8–11). Initial studies indicated that the
OSC-mediated prevention of chemically induced cancer correlat-
ed with induction of phase 2 carcinogen inactivating enzymes
and inhibition of cytochrome P450-dependent monooxygenases
(12–14).
More recent studies including those from our laboratory have
revealed that certain garlic-derived OSCs are effective in
suppressing proliferation of human cancer cells (15–25). The
OSC-mediated suppression of cancer cell growth in association
with G2-M phase cell cycle arrest and/or apoptosis induction was
documented for DADS and DATS in human colon, neuroblastoma,
and prostate cancer cells (15–20). Studies from our laboratory have
revealed that DATS is a much more potent suppressor of
proliferation of human prostate cancer cells compared with either
diallyl sulfide or DADS (18). Interestingly, a normal prostate
epithelial cell line is significantly more resistant to G2-M phase cell
cycle arrest by DATS compared with prostate cancer cells (19), a
feature highly desirable for cancer chemopreventive agents.
Mechanistic studies have revealed that DATS treatment not only
negatively regulates signaling pathways implicated in cell prolif-
eration (e.g., Akt) and angiogenesis (vascular endothelial growth
factor signaling axis) but also causes G2 and prometaphase arrest
and mitochondria-mediated apoptotic cell death (18, 19, 21–26).
Additionally, the growth of PC-3 human prostate cancer cells s.c.

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Cancer Research
implanted in athymic mice was significantly retarded by p.o.
administration of DATS (27).
Demonstration of in vivo efficacy of potential cancer chemo-
preventive agents in suitable animal models is a prerequisite for
their further clinical development. The present study was
undertaken to test whether DATS administration offers protec-
tion against prostate carcinogenesis in a transgenic mouse
model of prostate cancer (transgenic adenocarcinoma of mouse
prostate; hereafter abbreviated as TRAMP). We now show that
p.o. gavage of DATS significantly prevents development of
invasive prostate carcinoma and pulmonary metastasis multi-
plicity in TRAMP[C57BL/6FVB]F1 mice without causing weight
loss or affecting T-antigen expression.
Materials and Methods
Reagents. DATS (purity f99%) was purchased from LKT Laborato-
ries. Antibodies against CD31 (platelet/endothelial cell adhesion molecule
1; PECAM-1) and cyclinB1 were purchased from Santa Cruz Biotechnol-
ogy; the anti–E-cadherin antibody was from BD Transduction Laborato-
ries; the antibodies against proliferating cell nuclear antigen (PCNA) and
Ki-67 were from DakoCytomation; the anti-securin antibody was
purchased from MBL; and the antibodies against T-antigen and
synaptophysin were from BD Biosciences. The terminal deoxynucleotidyl
transferase-mediated dUTP nick-end labeling (TUNEL) staining was
performed using the In Situ Apoptosis Detection kit from Chemicon
International.
Animal model and DATS treatment. Various mouse models of
prostate cancer have been generated in recent years [e.g., TRAMP, LADY
(12T-10) transgenic mice, PTEN conditional knockout mice, etc.], which
develop lesions analogous to those observed in human prostate cancer
(reviewed in ref. 28). Our choice to use TRAMP mice for the present study
was based on the following considerations: (a) a rather well-defined
course of disease progression analogous to the human prostate
carcinogenesis from histologic PIN to invasive carcinoma with distant
site metastasis coupled with the overall high tumor incidence renders
TRAMP mice suitable for chemoprevention studies (29, 30), (b) the
chemoprevention studies in TRAMP mice can be completed in a
reasonable time frame (29, 30), and (c) TRAMP model has been used
successfully to test chemopreventive efficacy of several synthetic and
natural agents (31–33). We opted to use TRAMP[C57BL/6FVB]F1
progeny because these mice tend to form well-defined tumor nodules
and the frequency of phylloides-like structures, which are rare in human
prostate cancers, is relatively higher in C57BL/6 pure transgenic mice
(28–30). Male TRAMP[C57BL/6FVB]F1 hybrid mice were generated by
breeding female TRAMP in C57BL/6 background with nontransgenic FVB
male mice. Transgene verification was performed using DNA obtained
from tail clippings at 14 to 17 d of life as described by Greenberg and
colleagues (29). Use of mice for the studies described herein was
approved by the Institutional Animal Care and Use Committee. After
transgene verification, male TRAMP mice were maintained in a climate-
controlled environment with a 12-h light/12-h dark cycle. The mice were
fed food and water ad libitum. At age 8 wk, mice were randomized into
three groups. Mice in the control group (n = 16) received 0.1 mL PBS by
p.o. gavage thrice per week (Monday, Wednesday, and Friday), whereas
the experimental groups of mice (n = 19) received either 1 mg DATS/d or
2 mg DATS/d in 0.1 mL PBS by p.o. gavage thrice per week (Monday,
Wednesday, and Friday). Body weights of the control and DATS-treated
mice were recorded once each week beginning at the onset of the study.
After 13 wk of treatment, the animals were sacrificed 24 h after the last
administration of the vehicle or DATS by CO2 inhalation followed by
cervical dislocation. The weights of the vital organs, urogenital tract, and
prostate were determined. A portion of the prostate/tumor tissue was
placed in 10% neutral buffered formalin and paraffin embedded. Tissues
were sectioned at 4- to 5-Am thickness for H&E staining, TUNEL assay,
and immunohistochemical analyses.
Cancer Res 2008; 68: (22). November 15, 2008
Pathologic evaluation and scoring of tumor stage and metastasis.
Ten randomly selected fields on H&E-stained sections of the dorsolateral
prostate of individual mouse of control and DATS-treated groups were
independently scored by two investigators for incidence and percentage
of the area corresponding to each pathologic stage. Pathologic grading
was performed as described by Greenberg and colleagues (29, 30). Tissue
histology was classified as normal prostate gland with open ducts
lined with tall secretory epithelial cells surrounded by a closely
associated thin sheet of smooth muscle cells; PIN with piling up of
the epithelium, changes in the nuclear to cytoplasmic ratio, elongation
of the nucleus and an increase in epithelial stratification, and formation
of cribriform structures; well-differentiated (WD) carcinoma with clear
invasion of the epithelial cells into the stroma; and poorly differentiated
(PD) carcinoma with sheets of anaplastic cells with little or no glandular
structures. Lung, kidney, liver, and pelvic lymph nodes were evaluated for
the presence of metastasis by two independent investigators. The
incidence of metastasis (percentage of mice with metastatic lesions) in
each tissue and pulmonary metastasis multiplicity (number of pulmonary
metastatic lesions/mouse) were computed for the control and DATS-
treated groups.
Immunohistochemical analyses. Deparaffinized and rehydrated sec-
tions were quenched with 3% hydrogen peroxide and blocked with
normal serum. The sections were then incubated with the desired
primary antibody (anti–T-antigen, anti-PCNA, anti–Ki-67, anti–E-cadherin,
anti-synaptophysin, anti-CD31, anti-cyclinB1, or anti-securin antibody)
and washed with TBS followed by incubation with appropriate
biotinylated secondary antibody. Characteristic brown color was devel-
oped by incubation with 3,3-diaminobenzidine. The sections were
counterstained with Meyers Hematoxylin (Sigma) and examined under
a Leica microscope. At least three nonoverlapping representative images
of each tissue were captured from each section using a camera mounted
onto the microscope. The images were analyzed using Image ProPlus
5.0 software (Media Cybernetics) for quantitation of PCNA, Ki-67,
E-cadherin, T-antigen, cyclinB1, and securin expression and analysis of
microvessel number and vessel diameter (CD31 staining). Immunoblotting
for T-antigen expression using prostate/tumor tissue supernatants from
control and DATS-treated mice was performed as previously described by
us for other proteins (27).
Detection of apoptotic bodies by TUNEL staining. The paraffin-
embedded tissue sections were deparaffinized, rehydrated, and then used
to visualize apoptotic bodies by TUNEL staining using the ApopTag Plus
Peroxidase In Situ Apoptosis kit and following the manufacturer’s
protocol. Apoptosis was quantified by counting the number of TUNEL-
positive cells in at least three randomly selected, nonoverlapping high-
power fields.
Cytotoxicity of natural killer and dendritic cells. The natural killer
(NK) cells were isolated from the spleen of control and DATS-treated mice
using MACS beads (Myltenyi Biotech) as described by Giezeman-Smith and
colleagues (34) and cultured in interleukin-2–supplemented (1,000 units/
mL) medium. The DCs were isolated from the bone marrow of control and
DATS-treated mice using the adhesion selection method (35) and cultured
in medium supplemented with interleukin-4 (500 ng/mL), granulocyte
macrophage colony-stimulating factor (500 ng/mL), and Flt-3 (25 ng/mL).
After 5 d of culture, NK cells were plated in 96-well plates alone or
cocultured with dendritic cells (DC; day 5) at different ratios (10:0, 10:4, or
10:8). After 24 h, TRAMP-C1 target cells were added to the wells at specified
ratio. After 24 h, plates were spun down and supernatant was collected for
analysis of cytotoxicity using the Cytotox 96 nonradioactive assay
(Promega), which determines release of lactate dehydrogenase. Monolayer
cultures of TRAMP-C1 cells, a generous gift from Dr. Barbara Foster
(Roswell Park Cancer Institute, Buffalo, NY), were maintained as described
by us previously (36).
Statistical analysis. Statistical significance of difference in mean
urogenital and prostate weights and metastasis multiplicity between groups
was assessed by Wilcoxon test. The difference in metastasis incidence
between groups was assessed by Fisher’s exact test. Differences were
considered significant at P value of V0.05.
9504 www.aacrjournals.org

Results
P.o. administration of DATS prevented development of PD
prostate carcinoma in TRAMP mice. A preliminary dose-finding
study involving 8-week-old nontransgenic male [C57BL/6FVB]F1
littermates (5 mice per group) revealed that p.o. gavage of 1 and
2 mg DATS, thrice per week, was well-tolerated by the mice. The
nontransgenic mice treated with 1 and 2 mg DATS seemed healthy
and did not exhibit weight loss or signs of distress. All vital organs
(liver, lung, kidney, heart, and spleen) and prostate glands of 1 and
2 mg DATS-treated nontransgenic mice were also normal as judged
by histology and wet organ weight measurements (data not
shown). The DATS concentrations used in the present study (1 and
2 mg DATS) are within the range that can be generated through
dietary intake of garlic (37). As can be seen in Fig. 1A, the average
wet weight of the urogenital tract in mice given 1 and 2 mg DATS
was f26% and 35% lower, respectively, compared with control
mice. The average wet weight of the prostate gland in 1 and 2 mg
DATS-treated mice was also lower by f32% and 46%, respectively,
compared with control mice. The differences in urogenital and
prostate weights between control and DATS-treated mice did not
reach statistical significance due to large data scatter. However,
there was a trend of a dose-dependent decrease in average wet
weights of the urogenital tract and prostate in DATS-treated mice
compared with controls (Fig. 1A). As shown in Fig. 1B, the initial
and final body weights of the control and DATS-treated mice did
not differ significantly.
The histologic grades (normal prostate, PIN, WD carcinoma, and
PD carcinoma) in dorsolateral prostate of control and DATS-
treated mice with prostate weight of <1g were independently
scored by two investigators. Prostate sections from control as well
Figure 1. Effect of p.o. administration of 1 and 2 mg DATS, thrice weekly
(Monday, Wednesday, and Friday), on (A) urogenital tract and prostate weights
and (B ) initial and final body weights. Columns , mean (n = 16 for control group
and n = 19 for DATS treatment groups); bars , SE.
www.aacrjournals.org
DATS Inhibits Prostate Cancer Growth and Metastasis In vivo
as DATS-treated mice with prostate weight of >1 g were excluded
because the majority of the prostate gland in these mice was
occupied by PD carcinomas. Representative H&E staining in
dorsolateral prostate sections from two different control and
DATS-treated mice are depicted in Fig. 2A. The dorsolateral
prostate of the vehicle-treated control TRAMP mice exhibited low-
and high-grade PIN, WD carcinoma, and PD carcinomas along
with areas consistent with normal prostate. The incidence of the
PD carcinoma in the dorsolateral prostate of mice treated with 1
and 2 mg DATS was lower by 34% (P = 0.0147) and 41% (P =
0.035), respectively, in comparison with control mice (Fig. 2B).
Moreover, the area occupied by the PD carcinoma in the
dorsolateral prostate of DATS-treated mice was statistically
significantly lower compared with that in vehicle-treated control
mice. For example, the percentage of the area occupied by the PD
carcinoma in dorsolateral prostate of mice given 1 and 2 mg DATS
was lower by f64% (P = 0.0484) and 76% (P = 0.0189),
respectively, compared with control mice (Fig. 2C). The DATS
administration also resulted in a modest increase in the area
occupied by the PIN and WD carcinomas compared with control
mice (Fig. 2C). For example, the area occupied by the PIN in
dorsolateral prostate of mice treated with 1 mg DATS and 2 mg
DATS was higher by f57% (P = 0.0692 by Wilcoxon rank-sum test)
and 60% (P = 0.0277 by Wilcoxon rank-sum test), respectively,
compared with vehicle-treated control mice (Fig. 2C). Likewise, the
area occupied by the WD carcinoma in the dorsolateral prostate of
mice treated with 2 mg DATS was higher by f45% compared with
control mice (P = 0.0986; Fig. 2C). These results indicated that
DATS administration significantly inhibited progression from PIN/
WD to PD carcinoma in TRAMP mice, which was independently
verified by two investigators. These results are significant because
the mortality in prostate cancer patients is mainly attributable to
advanced and metastatic disease (2).
Prostate carcinogenesis in TRAMP mice is driven by the
expression of the viral large T and small t antigen in the secretory
epithelial cells of the prostate under the control of the minimal rat
probasin promoter (29). We considered the possibility that the
DATS-mediated prevention of PD carcinoma incidence/burden in
TRAMP mice was due to the suppression of the transgene
expression. We tested this possibility by determining the expression
of T-antigen in the prostate of control and 2 mg DATS–treated
mice by immunohistochemistry and immunoblotting. Fig. 3A
depicts immunohistochemical staining for the T-antigen expres-
sion in the prostate of a representative mouse of both control and
DATS-treated group. As can be seen in Fig. 3B and C, the DATS-
mediated inhibition of prostate cancer development in TRAMP
mice was not due to the suppression of the T-antigen expression.
DATS administration reduced pulmonary metastasis multi-
plicity. Because DATS administration significantly retarded the
development of PD carcinoma, we proceeded to determine the
incidence and multiplicity of pulmonary and pelvic lymph node
metastasis. Figure 4A depicts H&E staining in a lung section of a
representative mouse of both the control group and 2 mg DATS–
treated group. The incidence of pulmonary metastasis was f88%
in vehicle-treated control mice, which was reduced to f79% and
68% in mice given with 1 and 2 mg DATS, respectively (Fig. 4B).
Likewise, the incidence of metastasis to the pelvic lymph nodes was
f1.5- to 1.9-fold higher in the vehicle-treated control mice
compared with the DATS-treated mice (Fig. 4B). Majority of the
control mice exhibited multiple pulmonary metastatic lesions. The
multiplicity of the pulmonary metastasis in mice given 1 and 2 mg
9505 Cancer Res 2008; 68: (22). November 15, 2008
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Figure 2. A, H&E staining in the dorsolateral prostate of representative vehicle-treated control TRAMP mice and TRAMP mice given 1 or 2 mg DATS, thrice weekly
(Monday, Wednesday, and Friday) for 13 wk beginning at age 8 wk (magnification, 200). B, incidence of histologic grades classified as normal prostate gland (N ),
low- and high-grade PIN, WD carcinoma, and PD carcinoma in the dorsolateral prostate of vehicle-treated control TRAMP mice and TRAMP mice given 1 or 2 mg
DATS, thrice weekly (Monday, Wednesday, and Friday) for 13 wk. Tissue sections from mice with wet prostate weight of <1 gram were used because the majority of
the area of the prostate gland larger than 1 gram was occupied by the PD carcinoma. Ten representative fields of each section were independently scored by two
investigators. C, percentage of the area corresponding to the normal prostate gland, PIN, WD carcinoma, and PD carcinoma. Columns, mean (n = 11 for the control
group, and n = 15 and 13 for the 1 and 2 mg DATS treatment group, respectively); bars , variability of the results in histologic grading between two independent
investigators. *, significantly different compared with vehicle-treated control by Wilcoxon rank-sum test.

DATS was lower by f49% and 55% (P = 0.002) compared with
control mice (Fig. 4C). Together, these results indicated that DATS
administration delayed development of metastatic lesions espe-
cially pulmonary metastasis multiplicity in comparison with the
vehicle-treated control mice.
DATS administration decreased cellular proliferation and
synaptophysin expression in the dorsolateral prostate. Because
studies in cultured human prostate cancer cells (18, 25) and PC-3
xenografts (27) have shown that DATS treatment reduces cell
proliferation, we performed immunohistochemistry for well-
known proliferation marker PCNA (38) to determine the effect of
DATS treatment on proliferation index in the dorsolateral prostate
of TRAMP mice. Immunohistochemical comparisons for PCNA
expression in prostate of the control and DATS-treated mice were
carried out using size-matched tissues. Immunohistochemical
staining for PCNA expression in representative prostate of a
vehicle-treated control mouse and a 2 mg DATS–treated mouse is
shown in Fig. 5A. The PCNA expression was f46% lower in the
dorsolateral prostate of mice given 2 mg DATS compared with that
of control mice (P = 0.035). Inhibitory effect of DATS administra-
tion on proliferation index was confirmed by immunohistochem-
ical analysis of Ki-67 expression (Fig. 5B), which is another widely
used marker for cellular proliferation (39). The PD carcinomas in
TRAMP mice exhibit neuroendocrine (NE) differentiation (40, 41).
Because DATS administration inhibited incidence and burden of
PD carcinoma (Fig. 2B and C), we raised the question of whether
DATS treatment affected fraction of NE cells. We addressed this
question by determining the expression of synaptophysin in the

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 DATS Inhibits Prostate Cancer Growth and Metastasis In vivo

prostate of control and 2 mg DATS–treated mice. Synaptophysin is

a membrane-associated glycoprotein and well-accepted marker of
NE cells (41). The expression of synaptophysin in tissue sections
from control mice was predominant in the membrane (Fig. 5C).
The fraction of synaptophysin-expressing NE cells was significantly
lower in the prostate of 2 mg DATS–treated mice compared with
that of control mice. Collectively, these results indicated that the
DATS-mediated prevention of PD carcinoma development in
TRAMP mice correlated with reduced cellular proliferation and
suppression of NE differentiation.
Our previous studies in cultured human prostate cancer cells
have revealed that DATS treatment causes prometaphase arrest
that is characterized by accumulation of cyclinB1 and securin
proteins (21, 24). Consistent with the cellular results (21, 24), the
dorsolateral prostate of 2 mg DATS–treated mice exhibited
increased expression of cyclinB1 and securin proteins compared
with the control mice (Fig. 5D).
DATS administration failed to cause apoptosis or alter E-
cadherin expression. We have shown previously that DATS
treatment causes apoptosis in cultured human prostate cancer
cells (18, 22, 25). We performed TUNEL assay using prostate
sections from control and treated mice to test whether DATS-
mediated inhibition of prostate cancer development in TRAMP

Figure 4. A, H&E staining depicting metastatic lesion in the lung of a

representative control mouse and a 2 mg DATS–treated mouse (magnification,
200). B, incidence of metastasis in the lungs and pelvic lymph nodes of
vehicle-treated control TRAMP mice and TRAMP mice given 1 or 2 mg DATS.
C, pulmonary metastasis multiplicity in vehicle-treated control TRAMP mice and
TRAMP mice given 1 or 2 mg DATS. Columns , mean (n = 16 for the control
group and n = 19 for the DATS treatment groups); bars , SE. *, significantly
different (P < 0.05) compared with the vehicle-treated control group.

mice was due to increased apoptosis. Although the average number

Figure 3. A, immunohistochemical analysis for T-antigen expression in a
representative dorsolateral prostate of both control and 2 mg DATS–treated
mouse (magnification, 400). B, quantitation of T-antigen expression from
immunohistochemical analyses. Columns , mean (n = 4); bars, SE. C,
immunoblotting for T-antigen expression using lysates from prostate tissues of
three individual mice from both control and 2 mg DATS–treated groups. The blot
was stripped and reprobed with anti-actin antibody to correct for differences in
protein loading.
of TUNEL-positive apoptotic bodies was slightly higher in the
prostate of 2 mg DATS–treated mice compared with control mice,
the difference did not reach statistical significance (Fig. 6A).
E-cadherin is considered to be a suppressor of invasion and
growth of many epithelial cancers (42). Some anticancer agents
function by causing up-regulation of E-cadherin expression (32).
We therefore compared expression of E-cadherin in prostate of the

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Cancer Research
control and 2 mg DATS–treated mice. Expression of E-cadherin did
not differ in the dorsolateral prostate between control and 2 mg
DATS groups (Fig. 6B). Collectively, these results indicated that the
DATS-mediated prevention of PD carcinoma development in
TRAMP mice was not due to increased apoptosis or altered
expression of E-cadherin.
DATS administration failed to inhibit neovascularization.
We have shown previously that DATS treatment inhibits in vitro
angiogenic features (e.g., formation of capillary-like tube structures
and/or migration) in human umbilical vein endothelial cells and
prostate cancer cells (26). To test whether DATS administration
caused suppression of neovascularization in vivo, the dorsolateral
prostate sections from the vehicle-treated control and 2 mg DATS–
treated mice were stained for angiogenic marker CD31 (also known
as PECAM-1). The average number of vessels as well as the mean
vessel diameter did not differ significantly in the dorsolateral
prostate between the vehicle-treated control and the 2 mg DATS–
Cancer Res 2008; 68: (22). November 15, 2008 9508
treated mice (data not shown). These results indicated that the
DATS-mediated suppression of angiogenesis in vitro was not
translated into inhibition of neovascularization in vivo, at least
with the DATS dosing regimen used in the present study.
Cytotoxicity of NK and dendritic cells isolated from control
and DATS-treated mice against TRAMP-C1 target cells.
Experimental data exist to support the hypothesis that NK and
DC play an important role in immune surveillance during
tumorigenesis (43, 44). Moreover, garlic compounds have been
shown to modulate T-cell function (45). To test whether DATS-
mediated suppression of PD carcinoma development in TRAMP
mice was accompanied by boosting of NK/DC function, the
cytotoxicity of these cells isolated from control and 2 mg DATS–
treated mice was determined against TRAMP-C1 as a target cell
line. The DATS administration did not have any appreciable effect
on cytotoxic effects of NK cell alone or NK/DC cell cocultures
against TRAMP-C1 cells (data not shown).
Figure 5. Immunohistochemical analysis
for the expression of (A) PCNA
(magnification, 200), (B) Ki-67
(magnification, 400), and (C )
synaptophysin (600 for control and 400
magnification for 2 mg DATS–treated
group) in the prostate of a representative
mouse of both control and 2 mg
DATS–treated groups. The bar graphs
show quantitative data. Columns , mean
(n = 9–11); bars, SE. *, significantly
different (P < 0.05) compared with control.
D, immunohistochemical analysis for
cyclinB1 and securin protein expression in
the dorsolateral prostate of control and
2 mg DATS-treated mice. Columns , mean
(n = 3); bars, SE.
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 DATS Inhibits Prostate Cancer Growth and Metastasis In vivo

Figure 6. A, TUNEL staining in the dorsolateral prostate of a representative mouse of both control and 2 mg DATS-treated group (magnification, 200). The bar graph
represents quantitative analysis of TUNEL-positive apoptotic bodies/high power field. Columns , mean (n = 9–10); bars, SE. B, immunohistochemical analysis for
E-cadherin expression in the dorsolateral prostate of a representative mouse of both control and 2 mg DATS–treated group (magnification, 400). The bar graph
represents quantitative analysis of E-cadherin expression. Columns , mean (n = 9–10); bars, SE.

Discussion prometaphase cells (19, 21). The DATS-mediated G2 phase cell

The long latency of prostate carcinogenesis renders this disease
highly amenable to chemoprevention. Accordingly, identification
and preclinical evaluation of novel agents potentially useful for
chemoprevention of human prostate cancer is highly desirable and
could have a significant effect on disease-related cost, morbidity,
and mortality for a large segment of population. Guided by the
results of population-based case-control studies (6), we have
devoted considerable effort toward identification and preclinical
evaluation of Allium vegetable–derived sulfur compounds for their
efficacy against human prostate cancer cells (18, 19, 21–27). Our
previous studies have revealed that garlic constituent DATS is
highly effective in suppressing growth of human prostate cancer
cells in culture as well as in vivo in xenograft model (18, 19, 21–27).
The present study builds upon these observations and shows that
DATS administration prevents development of PD carcinoma and
multiplicity of lung metastasis in male TRAMP mice without
causing weight loss or affecting the T-antigen expression. The
incidence and the area occupied by the PD carcinoma were
statistically significantly lower in the dorsolateral prostate of
DATS-treated TRAMP mice compared with control mice. The
DATS concentrations effective against development of PD
carcinoma and pulmonary metastasis multiplicity are within the
range that can be generated through dietary intake of garlic (37). It
is important to point out that DATS has been administered to
humans at a dose of 200 mg in combination with 100 Ag selenium
every other day for 1 month without any harmful side effects (46).
We have shown previously that DATS treatment suppresses
growth of cultured human prostate cancer cells by causing G2 and
M phase cell cycle arrest (19, 21, 23, 24). Exposure of PC-3, DU145,
and/or LNCaP human prostate cancer cells to growth suppressive
concentrations of DATS results in accumulation of G2 and
cycle arrest in human prostate cancer cells is transient and
correlates with down-regulation and increased Ser216 phosphor-
ylation of cell division cycle 25C (19).4 On the other hand, the
prometaphase arrest resulting from DATS exposure seems
irreversible and persists for several hours even after removal of
the drug (24). The DATS-mediated prometaphase arrest is not
unique to the prostate cancer cells but accompanied by inhibition
of anaphase promoting complex/cyclosome as evidenced by
accumulation of its substrates cyclinB1 and securin (24). The
present study reveals that p.o. administration of DATS causes
growth arrest of cancerous cells in the dorsolateral prostate of
TRAMP mice. This conclusion is supported by the following
observations: (a) the dorsolateral prostates from DATS-treated
mice exhibit significantly lower protein levels of the proliferation
marker PCNA, a 36 kDa protein synthesized in early G1 and S
phases of the cell cycle and implicated in cell cycle progression,
DNA replication, and DNA repair (38); (b) DATS administration
causes a marked decrease in the protein levels of Ki-67, which is
a large nuclear protein preferentially expressed during all active
phases of the cell cycle (G1, S, G2, and M phase; ref. 39), in the
dorsolateral prostate; and (c) consistent with the results in cultured
human prostate cancer cells (24), the dorsolateral prostates from
DATS-treated mice display increased levels of cyclinB1 and securin
proteins. Thus, it is reasonable to conclude that reduced cellular
proliferation is an important mechanism in DATS-mediated
prevention of prostate cancer development in TRAMP mice.
We have shown previously that apoptosis induction is an equally
important mechanism in antiproliferative effect of DATS against
4
S.V. Singh, unpublished results.

www.aacrjournals.org 9509 Cancer Res 2008; 68: (22). November 15, 2008
Cancer Research

human prostate cancer cells (18, 25). It is interesting to note that
the number of apoptotic bodies is comparable in the dorsolateral
prostate of control and DATS-treated mice. Several possibilities
exist to explain discrepancies in the results between cultured
human prostate cancer cells and TRAMP model in vivo. One
possibility relates to the frequency and dose of DATS administra-
tion. A more intensive dosing regimen, such as higher dose and/or
daily administration of DATS, may be required to elicit apoptotic
response in the dorsolateral prostate of TRAMP mice in vivo.
Likewise, the possibility that earlier treatment with DATS (e.g.,
starting at age 4 weeks) leads to increased apoptosis as well as even
greater protection against prostate carcinogenesis in TRAMP mice
cannot be ignored. Additional work is needed to systematically
explore these possibilities.
Metastasis is the major cause of death in prostate cancer
patients (2). The pathogenesis of metastasis is dynamic and
complex involving a series of molecular events including synthesis
and secretion of several angiogenic factors to promote neo-
vascularization, motility and local invasion of the host stroma,
entry into the circulation, detachment, and extravasation (47).
Proliferation of the tumor cells within vasculature or the organ
parenchyma is necessary for the completion of metastasis process
(47). Steps leading to metastasis are complex and regulated by
multiple molecules including growth factors, matrix metallopro-
teinases, and cell adhesion molecules (47). For example, loss of
expression of cell adhesion molecules especially E-cadherin is
believed to be important for development of metastatic lesions
(42). Inhibition of prostate carcinogenesis and metastasis by plant
flavonoids apigenin in TRAMP mice was shown to correlate with
retained expression of E-cadherin (32). The present study reveals
that the DATS administration inhibits pulmonary metastasis
multiplicity in TRAMP mice. However, the inhibitory effect of
DATS administration against pulmonary metastasis multiplicity
seems independent of changes in E-cadherin expression or
inhibition of angiogenesis. Determination of the precise mecha-
nisms by which DATS administration inhibits pulmonary meta-
static multiplicity requires additional work.
The TRAMP model shares many features important in human
prostate cancer progression, including metastasis to distant sites,
progression to androgen independence, and NE differentiation (48).
The number of NE cells correlates with stage, Gleason Grade, and
survival in castration-recurrent prostate cancers (48–50). The PD
tumors and lymph node metastases in C57/BL6FVB TRAMP mice
express NE marker synaptophysin (40). We found that the fraction
of synaptophysin-expressing NE cells is significantly lower in the
prostate of 2 mg DATS–treated mice compared with control mice.
These results indicate that DATS administration suppresses NE
cells in TRAMP mice, which is consistent with inhibition of PD
carcinoma development.
In conclusion, the results of the present study indicate that p.o.
administration of DATS prevents development of PD carcinoma
and multiplicity of pulmonary metastatic lesions in TRAMP mice
without causing weight loss or affecting T-antigen expression. The
DATS-mediated prevention of prostate cancer development
correlates with reduced cell proliferation as evidenced by
suppression of PCNA and Ki-67 expression and accumulation of
cyclinB1 and securin proteins.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 5/5/2008; revised 7/21/2008; accepted 8/25/2008.
Grant support: National Cancer Institute grant CA113363. We thank Dr. Barbara
Foster for the generous gift of TRAMP-C1 cells.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.

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